Microencapsulation is a process by which a relatively thin coating can be applied to dispersions of small particles of solids or droplets of liquids, thus providing a means for converting liquids to solids, altering colloidal and surface properties, providing environmental protection, and controlling the release characteristics or availability of coated materials. Several of these properties can be attained by macropackaging techniques; however, the uniqueness of microencapsulation is the smallness of the coated particles and their subsequent use and adaptation to a wide variety of dosage forms and product applications. Heretofore, known feasible methods for producing microcapsules on an industrial scale have often involved the use of organic solvents. However, the use of organic solvents may present environmental and safety problems. In addition, it is often difficult to remove all the organic solvent from the microcapsules, thus leaving organic contaminants.
It has been proposed to use microcapsules as a means of delivering vaccine. Two broad types of antigen delivery systems have been studied for their capacity to enhance immunity: solid (or porous) microcapsules and microcapsules with a core region surrounded by a physically distinct wall. Solid microcapsules may be prepared by a variety of processes including coacervation of colloids (Kwok, K.K., et al., 1991, Pharm. Res. 8:341-344), precipitation of proteins by physical means (e.g., phase separation) (Santiago, N., et al., 1993, Pharm. Res. 10:1243-1247) or chemical agents (e.g., acid chlorides) (Levy, M. C., et al., 1991, J. Pharm. Sci. 80:578-585.), or solvent evaporation techniques that surround aqueous dispersions with polyester films (Singh, M., et al., 1991, Pharm. Res. 8:958-961). Wall/core systems shown useful for antigen delivery include liposomes (Gerlier, D., et al., 1983, J. Immunol. 131:490), ISCOMS (Claassen, I., and Osterhaus, A., 1992, Res. Immunol. 143:531-541) and proteosomes (Gould-Fogerite, S., and Mannino, R., 1992, Liposome Technology, Vol. III, Gregoriadis, G. (ed.), CRC Press, Boca Raton, Fla.; Miller, M. D., et al., 1992, J. Exp. Med. 176:1739-1744).
Perhaps the best studied of the antigen delivery systems are those derived from the linear polymeric esters of lactic acid and glycolic acid (i.e., poly (DL-lactide-co-glycolide)) (PLCG) (Edelman, R., et al., 1993, Vaccine 11:155-158; Eldridge, J. H., et al., 1989, Curr. Top. Microbiol. Immunol. 146:59-66; Eldridge, J. H. et al., 1990, J. Controlled Release 11:205-214; Eldridge, J. H., et al., 1989, Adv. Exp. Med. Biol. 251:191-202; Eldridge, J. H., et al., 1991, Mol. Immunol. 28:287-294; Eldridge, J. H., et al., 1991, Infect. Immun. 59:2978-2986; Marx, P. A., et al., 1993, Science 260:1323-1327; Moldoveanu, Z., et al., 1993, J. Infect. Dis. 167:84-90; O'Hagan, D. T., et al., 1993, Vaccine 11:149-154; O'Hagan, D. T., et al., 1991, Immunology 73: 239-242; Ray, R., et al., 1993, J. Infect. Dis. 167:752-755; Reid, R., et al., 1993, J. Immunol. 150:323A; Reid, R. H., et al., 1993, Vaccine 11:159-167). Encapsulation of putative antigens into PLCG microcapsules affords a number of advantages. First, microcapsules are easily degraded by hydrolysis to form lactic acid and glycolic acid. Second, PLCG microcapsules less than 5 .mu.m in size readily penetrate Peyer's patches, mesenteric lymph nodes and spleen after oral inoculation of mice. Third, oral, intraperitoneal, intranasal or subcutaneous inoculation of mice with PLCG microencapsulated antigens including influenza virus, parainfluenza virus, simian immunodeficiency virus, Staph. aureus enterotoxin B toxoid, and ovalbumin induces a greater immune response than that induced in animals inoculated with the same dose of free virus or protein. In addition, oral inoculation of mice with inactivated viruses induces an enhanced antigen-specific IgA response at mucosal surfaces. Lastly, PLCG microcapsules have been administered orally to adult volunteers without adverse effects.
The major disadvantage of PLCG microcapsules is the requisite use of organic solvents. Contact with organic solvents will inactivate the infectivity of viral and bacterial pathogens, and, in addition, may alter the immunogenicity of surface proteins critical to induction of humoral or cellular immune responses. In fact, large quantities of viral proteins have been required to induce an antigen-specific immune response with PLCG microcapsules.
Microencapsulation techniques are generally disclosed in U.S. Pat. No. 3,137,631; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat. No. 3,959,457; and U.S. Pat. No. 5,132,117. In addition, microencapsulation techniques are taught in Belgium Pat. 882,476 to Lim (1980); U.S. Pat. No. 4,744,933; and U.K. Pat. Appl. 2 135 954 A to Dautzenberg et al. (1984). However, these techniques have drawbacks, including the use of organic solvents or heat, which can lead to inactivation or denaturation of the antigen. More specifically, most methods for preparing immunogens such as vaccines in encapsulated form (e.g., PLGA's, cochleates, liposomes) require multiple, often harsh, processing steps (e.g., introduction of surface active agents, generation of liquid/liquid interfaces with different surface energies, dispersion in reactive organic liquid phases, mechanical shearing during emulsification and/or vortexing, heating to remove one or another volatile components). Each of the several processing steps can exert an adverse influence on functional integrity of some or all of the initial quantity of immunogen used in the encapsulation. Such a reduction in functional integrity of some fraction of the initial charge of immunogen is manifested in reduced stability and immunogenicity of the resulting formulation, and counters the desired enhancement of immune response sought through encapsulation. With agents of low immunogenicity, the toll taken by multiple harsh processing steps may defeat any benefits to be derived by encapsulation using methods already known in the art.
By contrast, International Publication WO 95/28227 describes a microencapsulation technology that utilizes an all-aqueous system. This technology is based on the formation of poorly soluble (amine) salts of polyanionic macromolecules. Using this technology, immunogenic compositions are encapsulated using an entirely aqueous system of reagents at or below room temperature and without need for high pressures. This process is capable of producing uniform size particles under very gentle conditions, and may be used to microencapsulate immunogenic compositions from infectious agents for use in vaccines.
An infectious agent of particular interest for encapsulation using the above-described all-aqueous system is bovine herpesvirus-1 (BHV-1), also known as infectious bovine rhinotracheitis virus. BHV-1 is a member of the alphaherpesviridae subfamily, and produces a variety of clinical forms of disease in cattle, including respiratory and genital infections, conjunctivitis, encephalitis, and abortions. Previous attempts at controlling BHV-1 infection have utilized vaccines comprising live attenuated virus (Gerber, J. D., et al., 1978, Am. J. Vet. Res. 39:753-760; Mitchell, D., 1974, Can. Vet. Jour. 15:148-151), inactivated virus (Frerichs, G. N., et al., 1982, Vet. Rec. 111:116-122), and viral subunits such as, e.g., one of the three major BHV-1 glycoproteins, which have been designated in the art as gI, gIII, and gIV (Babiuk, L. A., et al., 1987, Virology 159:57-66; van Drunen, S., et al., 1993, Vaccine 11:25-35). In addition, the ability of a recombinant, truncated version of the BHV-1 gIV glycoprotein (designated in the art as BHV-1 tgIV) to induce mucosal immunity against BHV-1 has been demonstrated (van Drunen, S., et al., 1994, Vaccine, 12:1295-1302). However, the immunoresponse to BHV-1 tgIV is low, requiring the administration of either large amounts of immunogen, which may be commercially impractical, or the addition of a potent adjuvant, which may in turn induce undesirable injection-site reactions. It was not known whether immunogenicity of a BHV-1 subunit component generally, and BHV-1 tgIV in particular, could be significantly enhanced by microencapsulation.